Electrostatic channeling of oxaloacetate in a fusion protein of porcine citrate synthase and porcine mitochondrial malate dehydrogenase

Mitochondrial malate dehydrogenase and citrate synthase are sequential enzy mes in the Krebs tricarboxylic acid cycle. We have shown [Lindbladh, C., Ra ult, M., Hagglund, C., Small, W. C., Mosbach, K., Bulow, L., Evans, C,, and Srere, P.A (1994) Biochemistry 33, 11692-11698] that a fusion protein of y east mitochondrial citrate synthase and yeast mitochondrial malate dehydrog enase channels oxaloacetate between the active sites. A Brownian dynamics s imulation model of porcine mitochondrial enzymes of citrate synthase and ma late dehydrogenase was used [Elcock, A. H., and McCammon, A. M. (1996) Bioc hemistry 35, 12652-12658], showing that a positive electrostatic surface po tential between the active sites of the fusion protein could account for th e channeling of oxaloacetate we observed with the yeast fusion protein. Sin ce the data were established with a yeast fusion protein and the model was with porcine fusion protein, we have now prepared and studied the porcine f usion protein, The channeling of the oxaloacetate intermediate was the same for the porcine fusion protein as it was for the yeast fusion protein. Thi s channeling behavior is eliminated at high ionic strength. A fusion protei n of porcine citrate synthase and porcine cytosolic malate dehydrogenase do es nor exhibit any channeling of oxaloacetate. A model of the fusion protei n with the cytosolic malate dehydrogenase shows no clear positive electrost atic potential surface between the two active sites, thus distinguishing it from the fusion protein with the mitochondrial malate dehydrogenase. These results establish the electrostatic nature of channeling in mitochondrial fusion proteins.